Hypoxic and endoplasmic reticulum (ER) stress are two essential components of the tumor microenvironment linking the cellular response to these factors with tumor growth. We have previously shown that hypoxia activates the unfolded protein response (DPR), a signaling pathway that is triggered in response to ER stress. X-box binding protein (XBP-1) is an important regulator of the transcriptional branch of this response and is spliced into its active for by IRE1, an ER transmembrane protein. Using XBP-1 deficient cells, we demonstrated that XBP-1 mediates survival under hypoxia and is critical for tumor growth. Given its role in regulating survival under hypoxia and its requirement for tumor growth, we hypothesize that targeting XBP-1 will be an effective therapeutic strategy. However, there are few examples of anti-cancer drugs that can effectively inhibit transcription factor activation. Therefore, our strategy for inhibiting XBP-1 is to block the activity of IRE1, an ER transmembrane protein responsible for splicing XBP-1 into its active form. IRE1 is activated by dimerization and autophosphorylation through its kinase domain. The endonuclease activity of IRE1 depends upon having an intact kinase domain, and to date, XBP-1 is the only described substrate for the endonuclease function of IRE1. In this proposal, we will investigate the effects of inhibiting XBP-1 using a pharmacologic and genetic strategy. We have identified a group of XBP-1 inhibitors (termed irestatins) in a high throughput screen of a 66,000 compound small molecule library. We have also generated several cell lines in which we inhibit XBP- 1 using dominant negative inhibitors of IRE and XBP-1 as well as a tetracycline regulated XBP-1 shRNA expressing cell line. We will monitor in vivo XBP-1 splicing activity within tumors using bioluminescent imaging of a luciferase reporter regulated by XBP-1 splicing. We will examine the effects of XBP-1 inhibition by multiple methods in a using a subcutaneous and orthotopic model of pancreatic cancer. We will use biochemical cell based assays to define the mechanism of action of the irestatins, investigate the consequences of XBP-1 inhibition on pancreatic tumor growth, and combine XBP-1 inhibition with other promising therapies for pancreatic cancer.